Genetic engineering bacteria producing beta- carotene and construction method of genetic engineering bacteria

Abstract

The invention discloses genetic engineering bacteria producing beta- carotene and a construction method of the genetic engineering bacteria. The construction method comprises the following steps of firstly knocking out alpha-glycerophosphate dehydrogenase gut2 genes in a glycolysis way in a beta-carotene synthetizing pathway in yarrowia lipolytica, then performing free expression on beta-hydroxyl-beta-methylglutaric acid erg13 genes having two endogenous copy numbers of the yarrowia lipolytica, and then performing integrant expression on the erg13 genes having two endogenous copy numbers of the yarrowia lipolytica at the position where the gut2 genes are knocked out; and performing free expression on endogenous hexokinase Hxk genes of the yarrowia lipolytica, and screening positive transformants to obtain the engineering bacterial strains producing beta-carotene yarrowia lipolytica. The bacterial strains are subjected to fermentation, culturing, extraction and separation, the beta-carotene is purified, and the content of the beta-carotene can reach 26.6mg / g cell dry weight.

Description

technical field [0001] The invention belongs to the technical field of metabolic engineering and combined biology, and specifically relates to a genetically engineered bacterium producing beta-carotene and a construction method thereof. Background technique [0002] β-carotene is a natural carotenoid found in green vegetables such as carrots and goji berries, and yellow and orange fruits. β-carotene, which is widely distributed in nature, mainly exists in the all-trans structure, containing multiple unsaturated conjugated polyene double bonds, which can undergo irreversible redox reactions with free radicals to achieve quenching of free radicals , anti-oxidation, inhibition of singlet oxygen, etc., and has anti-cancer, anti-cancer, anti-aging and other functions. From a structural point of view, one β-carotene molecule is equivalent to two molecules of vitamin A. As a nutritional pigment that has passed GRAS (the US FDA’s safety index for evaluating food additives), β-carot...

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Problems solved by technology

[0009] Although there have been many reports on the synthesis of β-carotene by Escherichia coli and Saccharomyces cerevisiae, there are still no related strains that have been applied to industrial production. The reasons may have the following points: (1) the key to β-carotene synthesis Enzymes (phytoene dehydrogenase, phytoene synthase / lycopene cyclase) have low activity, and the amount of carotene synthesis is not high after heterologous expression; (2) Escherichia coli will produce cells Toxin, there is a food safety risk when using it as an expression host to produce carotene; (3) β-carotene is a fat-soluble pigment, which is stored in liposomes, while Saccharomyces cerevisiae is a non-oleaginous yeast that accumulates The amount of lipids is less than 3% of the dry weight of the cells, which also limits the application of Saccharomyces cerevisiae as an expression host; therefore, increasing the production of liposomes helps to improve β-carotene, and it is possible to find a more advantageous microorganism Must do

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